Training Module For Water MICROBIOLOGY PDF

Food and Nutrition Research Institute
Department of Science and Technology
Training Manual
Microbiological Analysis of Drinking Water
This Training Manual was developed by the Food Analytical Service Laboratory (Laboratory Services
Group) of FNRI-DOST for the purpose of its training courses. This cannot be reproduced in partial or full
without the approval of FNRI.
Prepared by
Microbiology Unit
Food Analytical Service Laboratory (FASL)
Laboratory Services Group (LSG)
2013
2 Training in Microbiological Analysis of Water

Food Analytical Service Laboratory
TABLE OF CONTENTS
INTRODUCTION………………………………………………………………..………….4
(Course Description, Objectives and Mechanics)
TOPICS
Topic 1 Introduction to Water Microbiology………………………….…….....…..6

Topic 2 Microbiological Analysis of Water…………………………………....…10
LABORATORY OBSERVATION……………….……………………………………...….15
METHODS OF ANALYSIS
METHOD 1 Heterotrophic Plate Count ……………….…………….…18

METHOD 2 Total Coliform, Fecal Coliform and E.coli Count
by Most Probable Number Method…………………..…..22
APPENDICES
APPENDIX A WORKSHOP No. 1 Preparation and quality control of

culture media…………………………………..………….…28
APPENDIX B LABORATORY REPORT No. 1……………………………29
APPENDIX C WORKSHOP No. 2 Determination of quality of
drinking water from different sources .…….………..…….32
APPENDIX D LABORATORY REPORT No. 2…………………………....34
APPENDIX E LABORATORY REPORT No. 3……………………………35
APPENDIX F MPN TABLES…………………………………………….......36
APPENDIX G PHILIPPINE NATIONAL STANDARDS VALUE FOR
MICROBIOLOGICAL QUALITY OF WATER..……….…..38
APPENDIX H TRAINING SCHEDULE……………………………………..39
APPENDIX I POWERPOINT HANDOUTS…………………………….....40
APPENDIX J CURRICULUM VITAE.....................................................40

INTRODUCTION
Duration: Thirty (30) minutes
Learning Objectives
General: To discuss the course objectives, course content, significance of the
course, schedule of training and expected output.
Specific: After the session, the participants must be able to

1. enumerate the objectives of the course and its application;
2. give their expectations on the course and ensure that it will be
included in the training objectives;
3. evaluate their knowledge on water microbiology
Training Method: Lecture with visuals and discussion; Pre-evaluation (short quiz)
Materials Needed: Lecture presentation, blank CD, laptop computer and LCD
projector, office supplies and materials, white board marker and
eraser, pre-evaluation sheets, FNRI/FASL AVP
Content: Introduction / Overview of the Course
Course Description
This training on microbiological analysis of water covers introduction to the

course, requirements for water potability testing, proper sampling collection and
sample preparation, good laboratory practice (GLP) and laboratory quality
assurance (LQA), laboratory requirements, general principles and methodologies
in the conduct of microbiological analysis of water, and observation training on
the conduct of the analysis.
Training Objectives
This three day hands-on-training is intended to equip laboratory
managers/supervisors, technicians, analysts and other QA/QC personnel with
knowledge on water safety, water testing, and water quality through microbial
analysis.
Specifically, at the end of the training, the participants should be able to:
(a) explain the significance of microbiological analysis of water;
(b) enumerate and understand each test parameter included in water

potability testing;
(c) conduct proper sampling and handling of water samples for analysis;
(d) apply good laboratory practice and quality assurance in the laboratory;
(e) enumerate the laboratory requirements (e.g. equipment, facilities,

reagents, laboratory supplies), and the general principles of the test
method;
(f) conduct microbiological analysis of water in the laboratory; and
(g) demonstrate improvement in techniques on microbiological analysis of

water.
Training Schedule (Annex E)
Training Materials
1. LCD Projector
2. Lecture presentation/ materials
3. Blank CD
4. White Board/ White Board Marker/ Eraser
5. Sound System and Microphone (Lecture)
6. Laser pointer
7. Cassette recorder and blank cassette tapes for documentation
8. Office supplies and materials (bond paper, pens, pencils, etc.)
9. Equipment/facilities, reagents and lab. Supplies for the observation
training
10. Laboratory gown
Resource Persons / Trainers: see attached curriculum vitae

Topic 1. INTRODUCTION TO WATER MICROBIOLOGY
Duration: One (1) hour
Learning Objectives
General: To discuss basic knowledge on water microbiology
Specific: After the session, the participants should be able to:

1. discuss the importance of evaluating the microbiological quality
of water;
2. explain the health implications of microbial contaminations in
drinking waters;
3. enumerate the different indicators used in assessing the
microbiological quality of water; and
4. cite the existing standards in the country.
Training Method: Lecture with visuals and discussion
projector, blank cassette tapes and cassette recorder for
documentation, office supplies and materials, white board marker
and eraser.
Content: Water Microbiology
Clean Water is important to everyone however, still a significant number of

individual or population groups in the country have no or limited access to clean drinking
water (Hulton, et al., 2008). Unclean water supply may be the results of poor sanitation.
According to their report poor sanitation leads to an economic costs of about Php 77.8
billion per year
Over the years, there is still considerable number of outbreaks found to be

caused by drinking water, particularly in a developing country like ours. Worldwide,
statistics show that over 2 million people die every year due to waterborne pathogen
(Lechavallier & Buckley, 2007). In the Philippines, waterborne diseases rank among the
leading causes of mortality and morbidity (DOH, Philippines). Around 38 million cases of
diarrhea reported annually were attributed to poor sanitation and hygiene (Hulton, et al.,
2008) which includes access to unclean water.

Therefore it is important to evaluate the microbiological quality of water to protect
the consumers from illnesses due to consumption of water that may contain pathogens
such as bacteria, viruses, and protozoa.
Contagious diseases caused by pathogenic bacteria, viruses and parasites are

the most common and widespread health risks associated with drinking water.
However, routine examination for these pathogenic microorganisms is not
recommended except for investigations of water-related illness and special studies.
The major public health concern is that water should be free from contamination
by human or animal excreta, which can contain microbial contaminants. In order to
evaluate the quality of water, several indicators have been extensively studied and
these remain the most sensitive and specific way of assessing the hygienic quality of
water. These are the microorganism(s) used as indicators for monitoring according to
the WHO guidelines (2011):
 E.coli
E.coli is the most suitable indicator of fecal contamination. It is used in monitoring
programs such as surveillance of drinking water quality. Detection of which
requires further sampling and investigation of potential sources.
 Total coliforms
Total coliforms should be absent immediately after disinfection and presence of
these organisms indicates inadequate treatment.
 Heterotrophic Plate Count
HPC is useful in operational monitoring as a treatment and disinfectant indicator.
It is also valuable in assessing cleanliness and integrity of distribution systems
and detecting presence of biofilms
 Clostridium perfringens
The spores of C. pefringens is known to be exceptionally resistant under
unfavorable conditions – the reason why it has been proposed as an indicator of
protozoa in drinking water supplies. Presence of this organism indicates
intermittent fecal contamination and requires further investigation.
 Intestinal Enterococci
These can be used as indicators of fecal contamination since these organisms
are excreted in the feces of human and warm blooded animals. Detection of
which should also lead to further action including investigation.
 Coliphages
These are viruses that only use bacteria (specifically E.coli and other related
genera) as hosts for multiplication. They typically replicate in the gastrointestinal
tract of humans and other warm blooded animals. Their presence in drinking

water signifies presence of fecal pollution and potential presence of enteric
viruses and other pathogens.
 Bacteroides fragilis phages
These are inhabitants of the human gastrointestinal tract in greater numbers
compared to E.coli. Its presence is a sound evidence of fecal contamination as
well as inadequacy in the water treatment and disinfection process.
 Enteric viruses
The presence of these viruses, which infect the human gastrointestinal tract and
are largely transmitted by fecal-oral route, is a certain evidence of fecal pollution
however practical methods for routine monitoring of water supplies for these
organisms are not yet available.
Standard methods for the detection of the above-mentioned organisms are being
used in the routine examinations of water quality.
It is important to take note that criteria for microbial quality of water should be
uniform in different laboratories and internationally. In the Philippines, National
Standards for Drinking Water was issued in 2007 by the Department of Health under the
Administrative Order No. 2007-0012. It aims to protect the public health, safety, and
welfare by ensuring the quality standards of drinking water.
References
American Public Health Association – American Water Works Association

Standard Methods for Examination of Water and Wastewater. 21st edition.
2005.
Figueras, M.J. & Borrego, J.J. (2010). New Perspectives in Monitoring Drinking
Water Microbial Quality: A Review. International Journal of Environmental
Research and Public Health, 7, 4179- 4202. doi: 10.3390/ijerph7124179
Hulton, G, Rodriguez, UE., Napitupulu, L., Thang, P., Kov, P. (2008). Economic
impacts of sanitation in Southeast Asia. Worldbank Water and Sanitation
Program.
Lechavallier, M. & Buckley, M. (2007). Clean Water: What is acceptable microbial

risk. Retrieved from the American Academy of Microbiology website:
http://academy.asm.org/index.php/water/427-clean-water-what-is-acceptable-microbial-
risk

Philippines Department of Health. (2011). Provision of Potable Water Program
(SALINTUBIG Program – Sagana at Ligtas na Tubig para sa lahat) Retrieved
from DOH website: http://www.doh.gov.ph/content/provision-potable-water-program-
salintubig-program-sagana-ligtas-na-tubig-para-sa-lahat.html
Philippines Department of Health. (9 Mar 2007). Philippine National Standards

for Drinking Water (Administrative order No. 2007-0012). Retrieved from
DOH website: http://recordsvr.doh.gov.ph/appnet/public/p/ai/searchall.htm
World Health Organization. (2011). Chapter 5: Surveillance. Guidelines for

Drinking Water Quality (4th ed.) Malta: Gutenberg.
World Health Organization. (2011). Chapter 7:Microbial Aspects. Guidelines for

Drinking Water Quality (4th ed.) Malta: Gutenberg

Topic 2 MICROBIOLOGICAL ANALYSIS OF WATER
Duration: One (1) hour lecture, One (1) hour laboratory
Learning Objectives
General: To discuss the general procedures in the microbiological analysis of
water
1. enumerate and understand each of the steps prior to the
microbiological analysis of water;
2. understand the requirements of the different methods;
3. prepare the materials for the analysis;
4. apply appropriate techniques; and
5. perform microbial analysis using standard or validated methods.
Training Method: Lecture with visuals and discussion; Demo on sample collection of
drinking water
Workshop no. 1 – Preparation and quality control of culture media.
projector, blank cassette tapes and cassette recorder for
documentation, office supplies and materials, white board marker
and eraser.
Content: Microbiological Analysis of water
In assessing the microbial quality of water, it is very important that laboratory

examinations are done with the representative samples of water taken at all critical
stages of the water supply. Microbiological examination is conducted more frequently
because of the high probability of microbial contamination and the extent of public
health it might cause.
It is also significant to learn the basic techniques found in the Compendium of

methods for the microbiological examinations of food (2001) such as:
 Dilution Technique
 Plating Methods
 Most Probable Number Method

There are different steps involved in the analysis of water. These are as follows:
1. Media Preparation
Prior to the actual analysis of sample, culture media, reagents, and other
materials should be prepared beforehand.
2. Sample Collection
Water samples taken to the laboratory should be a representative of the
water being examined. The Administrative Order No. 2007-0012 of the
Department of Health issued the Philippine National Standards for Drinking
Water 2007, wherein it includes the guidelines for sampling point selection in its
Annex B.
In collecting samples, non-reactive borosilicate glass or plastic bottles must
be used. These containers should be cleansed and rinsed carefully with
deionized or distilled water, and then sterilized.
Detailed instructions on the collection of samples for bacteriological analysis
are given in Standard Methods for the Examination of Water and Wastewater
(APHA et al., 2005). To avoid unpredictable changes in the bacterial flora of the
sample, examination should be started as soon as possible after collection. The
sample should be transported to the laboratory in a cooler containing ice (at 5 ±
3°C), to minimize changes in populations and concentrations. Holding time of
up to 30 hours must not be exceeded for the analysis of coliforms and 8 hours
for the HPC (APHA et al., 2005).
Samples should be labeled with the following information along with the
identification number linked to the sample bottle and is recorded on
accompanying forms:
a. Time
b. Date
c. Location
d. type of sample (e.g., raw water, distribution system)
e. sampler’s name, and identification number (if used)
f. disinfectant residual measurements, and
g. any special conditions.
When examination will be delayed, it is particularly important to record the

duration and temperature of storage, as this information should be taken into
consideration when interpreting the results.
3. Inoculation
4. Incubation

5. Reading of results
6. Computation and Reporting of Results
In the conduct of microbiological analyses of water, validity of analytical data are

also important. Establishment of a Quality Assurance Program is required to minimize
errors. The following are the intralaboratory quality control guidelines from the Standard
Methods for the Examination of Water of Wastewater (2005) that must be addressed by
laboratories according to their specific needs and planned use of the data:
1. Personnel
 The analyst should be adequate in number, qualified and trained to do the
analysis. Training record should include evaluation of competency.
 The supervisor is responsible for the periodic review of IQC data, and
working procedures to identify gaps and minimize occurrence of errors.
 Microbiological testing should be performed by a professional
microbiologist or a technician trained in environmental microbiology, if
possible.
According to the Philippine Accreditation Office (LA/SR02,2009) minimum
requirement for a competent analyst is to be a graduate of microbiology, food
science, pharmacology, biotechnology, biochemistry, toxicology, veterinary
science and medical technology.
2. Facilities
Laboratories should be well-ventilated, designed to utilize the spaces
available, provide linear bench spaces for analysts’ activities, be
thoroughly cleaned, maintained and monitored.
3. Laboratory Equipment and Instrumentation

 It must be verified that each equipment meets the user’s needs for
precision and accuracy
 Perform equipment maintenance on a regular basis
 Record all data in a permanent log book or form
4. Laboratory Supplies
 Glassware should be examined before use
 Materials should be of proper material
 Reagents, dyes, stains, and culture media should be assured of quality
prior to use

5. Standard Operating Procedures
 SOPs should describe in detail all laboratory operations

 They should be unique to the laboratory
 These guide routine operations, help assure uniform operations, and
provide a solid training tool.
6. Analytical Methods
 Microbiological methods used and followed in the laboratory should be
standardized to produce uniform results from multiple laboratories
 Should be available to each analyst
 Should be validated/verified and be appropriate to each sample analyzed
7. Analytical Quality Control Procedures

QC must be performed in each analysis to ensure validity of results.
These include:
o Control cultures
o Duplicate analyses
o Sterility checks
8. Verification
Methods should be verified/confirmed for different water types and
different methods
9. Documentation and Recordkeeping

 It is important that all data and procedures are properly documented and
that the laboratory has a Quality Manual and SOP ensuring that the
objectives of the management will be achieved.
 Records of microbiological analyses should be kept for at least 5 years
From the previous discussion, it was mentioned that there are a lot of
microbiological contaminants present in water as well as indicators used to assess its
overall hygienic quality. The Standard Methods for Examination of Water and
Wastewater lists different methods for several microorganisms that are found in water.
These organisms are the causative agents of several waterborne disease outbreaks.
However, the more practical approach is to examine the water for indicator organisms
specifically associated with fecal contamination as a routine.

The standard parameters for water testing include: Heterotrophic Plate Count,
Total Coliform Count, and Fecal Coliform Count. Refer to Annex D for the Standard
Methods of Detection and Values for the Microbiological Quality.
References
Downes, F. & Ito, K. (2001). Compendium of Methods for the Microbiological

Examination of Foods. (4th ed). Washington, DC: American Public Health
Association.
Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (2005). Standard Methods
for Examination of Water and Wastewater. (21st edition). Maryland, USA:
American Public Health Association.
Philippines Accreditation Office (December, 2009). Supplementary Requirements for

Accreditation in the Field of Biological Testing. LA/SR02 Issue No. 2.
Philippines Department of Health. (March 9, 2007). Philippine National Standards for

Drinking Water (Administrative order No. 2007-0012). Retrieved from DOH
website: http://recordsvr.doh.gov.ph/appnet/public/p/ai/searchall.htm
World Health Organization. (2011). Guidelines for Drinking Water Quality. (4th ed).
Malta: Gutenberg.

LABORATORY OBSERVATION
MICROBIOLOGICAL ANALYSIS OF DRINKING WATER
Duration: Fifteen (15) hours of lecture and laboratory
Learning Objectives
General: To discuss the techniques and quality control procedures employed

in Heterotrophic Plate Count (HPC) and Total Coliform, Fecal Coliform
Count, and E.coli Count
1. enumerate the techniques and precautions during analysis;
2. identify the quality control procedures employed in the analysis;
3. conduct microbial analysis in the laboratory: HPC, Total
Coliform/Fecal Coliform/Ecoli Count; and
4. apply and implement quality control procedures in the
laboratory.
Training Method
Observation training in the Food Analytical Service Laboratory on microbiological

analysis of water.
Method 1 – Heterotrophic Plate Count: Pour plate Method
Method 2 – Total Coliform Count, Fecal Coliform and E.coli Count: MPN Method
Workshop No. 2 – Determination of quality of drinking water from different

sources.
Materials Needed
Laboratory supplies, Culture media/reagents, equipment for microbiological

analysis, notebook, calculator, laboratory gown, Working Reference Cultures

METHODS
OF
ANALYSIS

CONTENTS
Method 1 – Heterotrophic Plate Count

(Pour plate method)
Method 2 – Total Coliform / Fecal Coliform /

E.coli Count
(MPN Method)

Method 1
HETEROTROPHIC PLATE COUNT (Pour plate method)
1. PURPOSE/SCOPE:
This method provides an approximate enumeration of the total number of viable

bacteria in a given water sample, which yields useful information about water
quality and may provide supporting data on the significance of the coliform
results. This is useful in judging the efficiency of various treatment processes and
may have significant application as an in-plant control test.
2. SAFETY:
2.1 Perform method in aseptic conditions, using Biological Safety Cabinet

(Class II).
2.2 Wear proper laboratory attire: laboratory gown, mask, hair cap, gloves,
closed shoes.
2.3 Delay in pouring of agar after dilution and dispensing may lower counts for
several reasons while increased holding time to dilutions leads to higher
counts.
2.4 Culture medium should be cooled at 450C prior to use.
3. REFERENCE DOCUMENTS:
Standard Methods for Examination of Water and Wastewater. 21st edition. 2005.
American Public Health Association – American Water Works Association.
4. DEFINITION:
Heterotrophic Plate Count includes all microorganisms that are capable of

growing in or on a nutrient rich solid agar medium.
5. PRINCIPLE:
Heterotrophic Plate Count may be determined by pour plate method, spread

plate method or membrane filtration method. In this exercise, pour plate method
will be used. It makes use of Plate Count Agar as the culture medium. Colonies
arise in pairs, chains, clusters, or single cells, all of which are included in the term
“colony forming units” (CFU).

6. CULTURE MEDIA/ MATERIALS:
All media shall be of recognized quality. The reagent water used shall be distilled
water. Note: Culture media and reagent water should undergo quality control
check (intermediate) before use.
6.1 Plate Count Agar

6.2 Bactopeptone Water
7. EQUIPMENT/APPARATUS:
7.1 Biosafety Cabinet

7.2 Incubator, 350C
7.3 Water bath, 450C
7.4 Sterile pipettes
7.5 Pipette aid and Pipettor
7.6 Diluent bottles
7.7 Sterile plates
7.8 Refrigerator (0 to 40C)
7.9 Colony Counter
8. PROCEDURE:
8.1 Dilution and Inoculation
8.1.1 Collect water sample as described in ANNEX B. Initiate analysis as

soon as possible.
8.1.2 Thoroughly mix the water sample by making complete 25 back and forth
movements.
8.1.3 Prepare dilution by transferring 1.0 ml from the water sample to the 99
ml diluent. Shake dilution vigorously.
8.1.4 Inoculate 1.0 ml of water sample to properly marked, duplicate plates.

This serves as 100 dilution. Also, inoculate 0.1 ml from the same
sample to duplicate plates (This is the 10-1 dilution).
8.1.5 From the 99 ml diluent mixed with 1 ml of the original water sample,
inoculate also 1.0 ml and 0.1 ml volumes each to duplicate plates,
labeled as 10-3 and 10-4, respectively.

8.1.6 Pour 15 ml of Plate Count Agar to the plates and mix carefully to evenly
distribute the colonies. Make sure that the agar is tempered to 45 to
460C using a thermostatically controlled water bath to avoid heat
shock to bacteria.
Note: Use sterile pipette for initial and subsequent transfers from each
container; and separate sterile ones from each dilution. Also, do not let 20
minutes elapse between starting pipetting and pouring plates.
8.2 Incubation
8.2.1 Let agar solidify and invert plates for incubation.
8.2.2 Incubate plates at 350C for 48 hours.
8.3 Counting and Recording of Results
8.3.1 Colonies produced are relatively small and compact. Count all colonies
on selected plates promptly after incubation. Consider plates with
30 to 300 colonies each.
8.3.2 If there is no plate with 30 to 300 colonies, and one or more plates have
more than 300 colonies, use the plate(s) having a count nearest to
300 colonies.
8.3.3 If the number of colonies per plate far exceeds 300, do not record as
TNTC (too numerous to count), refer to discussion in the
powerpoint.
8.3.4 If there are spreading colonies, count colonies on representative

portions only when colonies are well distributed in spreader-free
areas and the areas covered by the spreaders does not exceed
one half of the plate area.
8.3.4.1 If spreading colonies must be counted, refer to the discussion in

the powerpoint.
8.3.4.2 If plates have excessive spreader growth, record as

“SPREADERS” (SPR).

8.3.5 When plates are uncountable because of missed dilution, accidental
dropping, and contamination, or the control plates indicate that the
medium or other material or labware was contaminated, report as
“LABORATORY ACCIDENT” (LA).
9. CALCULATION OF RESULTS:
9.1 Use this formula:
N = average number of colonies
Actual Volume of Sample in dish, ml
9.2 Report test results to two significant digits
10. REPORTING OF RESULTS:
10.1 Report results as CFU per ml.
10.2 In cases of 8.3.2 and 8.3.3, report results as estimated CFU/ml
10.3 If plates from all dilutions of any sample have no colonies, report the count
as less than one (< 1) divided by the corresponding largest sample volume
used. Report as estimated CFU/ml.
10.4 Report result as greater than (>) 6500, for glass plates, or (>) 5700, for
plastic plates, when bacterial plates on crowded plates are greater than
100 colonies /cm2. Report as estimated CFU/ml.
11. RECORDS:
11.1 Sample Worksheet (Annex D & E)

Method 2
TOTAL COLIFORM COUNT, E.coli COUNT, AND FECAL COLIFORM
(MPN method)
1. PURPOSE/SCOPE:
This method aims to estimate the mean density of coliforms, and other
organisms, in the sample. The technique uses presumptive-confirmed phases or
completed test. For routine examination of public water supplies, the total
coliform test determines the efficiency of treatment plant operations and integrity
of the distribution system. This method also allows distinction of fecal coliforms
from the total coliforms using EC medium. And most importantly, this procedure
allows detection and/or enumeration of Escherichia coli from the water sample
which indicates fecal contamination and possible presence of enteric pathogens.
2. SAFETY:
2.1 Wear proper laboratory attire (laboratory gown, hand gloves, facial mask,
hair cap) to protect self
2.2 All analyses are to be carried out in an operating biological safety cabinet
(Class II)
2.3 Maintain control cultures
2.5 All equipment and work benches should be disinfected routinely.
3. REFERENCE DOCUMENTS:
Standard Methods for Examination of Water and Wastewater. 21st edition. 2005.
American Public Health Association – American Water Works Association.
4. DEFINITION:
Multiple-tube Fermentation Technique is a standard test for the coliform group
Coliform group of bacteria (also called as total coliforms) is defined as all the
aerobic and facultative anaerobic, gram negative, nonspore forming, rod-shaped
bacteria that ferment lactose, with gas formation within 48 hours at 35 0C.
Fecal Coliforms or the thermotolerant fecal coliforms are a subgroup of total

coliforms that are differentiated from the total coliforms through laboratory
examinations using elevated temperatures (43 to 44.50C).

Escherichia coli is most common facultative bacterium in the intestines of warm
blooded animals. This is the indicator of choice for fecal contamination.
5. PRINCIPLE:
MPN method or referred as Multiple-tube Fermentation technique in SMEWW

utilizes replicate tubes and dilutions and results are reported in terms of Most
Probable Number (MPN) of organisms present. This number is based on certain
probability formulas. Bacterial density is estimated from the table using the
number of positive tubes in the multiple dilutions.
6. CULTURE MEDIA/ MATERIALS:

All media shall be of recognized quality. The reagent water used shall be distilled
water. Note: Culture media and reagent water should undergo quality control
check (intermediate) before use.
6.1 Lauryl Tryptose Broth

6.2 Brilliant Green Lactose Bile Broth
6.3 EC medium
6.4 Bactopeptone Water
6.5 Tryptone water
6.6 Kovac’s reagent
7. EQUIPMENT/APPARATUS:
7.1 Biosafety Cabinet

7.2 Incubator, 350C
7.3 Water bath, 44.50C
7.4 Sterile pipettes and pipette aid
7.5 Pipette aid and Pipettor
7.6 Diluent bottles
7.7 Sterile tubes
7.8 Inoculating loop and loop sterilizer
7.9 Vortex mixer
8. PROCEDURE:
8.1 Inoculation for Presumptive Testing
8.1.1 Collect water sample (100 ml) as described in Topic 2. Initiate analysis
as soon as possible.
8.1.2 Thoroughly mix the water sample by making complete 25 back and forth
movements.
8.1.3 Arrange fermentation tubes containing Lauryl Tryptose Broth (with
Durham tubes inside) in rows of five or ten tubes each in a test tube
rack. Prepare dilutions as described in Method 1. Shake dilutions
vigorously.
For Potable Water, any of these can be used:

 Five 20-ml portions
 Ten 10-ml portions
 Single bottle of 100 ml portion
8.1.4 Inoculate each tube in a set of five with replicate sample volumes (in
increasing decimal dilutions). Mix test portions in the LST tubes
gently.
Note: Use sterile pipette for initial and subsequent transfers from each
container; and separate sterile ones from each dilution. Also, do not let 20
minutes elapse between starting pipetting and pouring plates.
8.1.5 Incubate inoculated tubes or bottles at 35 0C. Observe tubes after 24

hours for growth and gas production. If no gas is evident,
Reincubate and reexamine at the end of 48 hrs. Record presence
of growth and gas as positive presumptive reaction.
8.2 Confirmatory testing for Total Coliforms
8.2.1 All presumptive tubes showing growth and/or gas are subject to
confirmatory testing.
8.2.2 Gently shake tubes and using a sterile loop, transfer one or more loopful
of culture to a fermentation tube containing Brilliant Green Lactose
Bile Broth (with Durham tube). Repeat for all other positive
presumptive tubes.
8.2.3 Incubate the inoculated BGLB tubes at 35 0C and observe for gas
production in the Durham tube within 48 hours.
8.2.4 Record the positive tubes for the computation of MPN value in 9.1.

8.3 Confirmatory Testing for Fecal coliforms
8.3.1 All presumptive tubes from 8.1.5 will be used as well for Fecal Coliform
Testing.
8.3.2 Gently shake tubes and using a sterile loop, transfer one or more
loopful of culture to a fermentation tube containing EC Medium
(with Durham tube). Repeat for all other positive presumptive tubes.
8.3.3 Incubate the inoculated EC tubes at 44. 50C (water bath incubator) for
24 hours and observe for gas production in the Durham tube.
Failure to produce gas constitutes a negative reaction.
8.3.4 Record the positive tubes for the computation of MPN value in 9.1.
Note: Place all EC tubes in water bath within 30minutes after inoculation.
Maintain sufficient water depth in water bath incubator to immerse
tubes to upper level of the medium.
8.4 Completed Test for E. coli
8.4.1 All presumptive tubes from 8.1.5 will be used also for Escherichia coli
Testing.
8.4.2 Gently shake tubes and using a sterile loop, transfer one or more loopful
of culture to a tube containing 5 ml of Tryptone water. Repeat for
all other positive presumptive tubes.
8.4.3 Incubate inoculated tryptone water in a water bath or incubator

maintained at 44.5 0C for 24 hours.
8.4.4 After incubation, add 0.2 to 0.3 ml Kovac’s reagent to each tube of
tryptone water.
8.4.5 Examine all tubes for the presence of a deep red ring in the upper layer.
Presence of red color indicates a positive response for E.coli.
8.4.6 Record the results of the indole-positive tubes and use for the
calculation of MPN in 9.1.

9. CALCULATION OF RESULTS:
9.1 Use the values obtained from 8.2.4, 8.3.4 and 8.4.6 to compute for the MPN
values.
9.2 Refer to Annex F for the appropriate MPN tables to use. Refer to the
Exercises Discussion on MPN for reporting accurately.
10. REPORTING OF RESULTS:
Report results as MPN/100 ml
11. RECORDS:
Sample Worksheet (Annex D & E)

ANNEX
WORKSHOP No. 1 Preparation and quality control of
A culture media
B LABORATORY REPORT No. 1
C WORKSHOP No. 2 Determination of quality of

drinking water from different sources
D LABORATORY REPORT No. 2
E LABORATORY REPORT No. 3
F MPN TABLES
G PHILIPPINE NATIONAL STANDARDS VALUE FOR

MICROBIOLOGICAL QUALITY OF WATER
H TRAINING SCHEDULE
I POWERPOINT HANDOUT
J CURRICULUM VITAE

ANNEX A
WORKSHOP No. 1
PREPARATION AND QUALITY CONTROL OF CULTURE MEDIA
Cultivation of bacteria (i.e., their growth on a nutrient medium) is necessary for subsequent
isolation and identification. In this exercise, participant will prepare the necessary media for testing
potability of drinking water including broth tubes, agar slants, agar deeps, and agar plates. Different
performance test will be use to ensure that the media prepared is of good quality and capable of
giving satisfactory results. The culture medium will be evaluated for its sterility, physical
characteristics, productivity, selectivity and biochemical reactions after inoculation with target or
non-target microorganisms and incubation to desired temperature.
MATERIALS
Culture (24-hour broth) Miscellaneous supplies

Escherichia coli Aluminum foil/weighing boat
Staphycoccus aureus Spatula
Salmonella typhimirium Stirring bar
Enterobacter aerogenes Bunsen burner/bacticinerator
Distilled water
Media Erlenmeyer flask, 250 ml
Graduated cylinder, 100 & 500 mL
Equipment Inoculating loop and needle
Autoclave Petri dishes, sterile
Balance Pipette, 10 ml
Hot plate Test tubes and caps
Waterbath (set at 44.5oC) Test tube rack
Incubator (set at 35°C)
PROCEDURE
Prepare a list of all media that will be used in the analysis of drinking water. Classify the
media according to its purpose/selectivity. Determine the required performance test/quality
assurance test for each media listed. Prepare the media using these basic steps as guide.
1. Follow the directions on the media bottle. Weigh carefully the proper amount of
dehydrated medium
2. Place the requisite amount of distilled water into a suitable container
3. Add the weighed dehydrated medium to part of the water. Mix
4. Add the remaining water and mix again.
5. Check pH and adjust if necessary
6. Heat to boiling to complete dissolution using microwave, hot plate or water bath.
7. Stir often to prevent overheating and burning
8. Distribute medium to appropriate containers, making sure that the amount of medium
per container is no more than 2/3 of the containing volume of the container.
9. Sterilize at 1210C for 15 minutes or according to the recommended procedures of the
medium.
10. Melt and hold media at 44 to 460C until ready to use, but not exceeding 3 hours.

ANNEX B
LABORATORY REPORT No. 1
PREPARATION AND QUALITY CONTROL OF CULTURE MEDIA
Name:__________________________________ Date:________________
A. Preparation of media and inoculation of growth media.
Use the table below to design quality control of media for potability test of drinking water.
Test Culture Media Selectivity Performance Acceptance

Test Criteria
LST Broth Non selective Productivity >70% recovery of
target organism
Total BGLB
Coliform Count
Fecal Coliform
Count
E. coli
Count
Source:
Standard Methods for the Examination of Dairy Products
American Public Health Association
Chapter 4: Media and Dilution Water Preparation

B. SAMPLE MEDIA PREPARATION LOGSHEET
Record the weight and volume of each commercially prepared media used and complete
the form for MEDIA PREPARATION and QUALITY CONTROL. Use appropriate control strains
for growth performance testing of laboratory prepared
Volume Sterility Control
Amount of
Name of Lot Desired
Test Medium
Brand
Number
weighed water pH Room
(g) used incubation + -
Temp
(ml) Temp
BPW
Coliform/
Fecal Coliform/
E. coli
Heterotrophic
Plate Count

C. Answer the following questions in the space provided.
1. Why is it essential that media be sterile prior to use?
2. Why must agar be cooled prior to pouring plates?
3. Why are the inoculating loop and needle flamed before and after use?
4. What is contamination? What are the possible contaminants in the lab?
5. How does one determine if growth has occurred in broth?

ANNEX C
WORKSHOP No. 2
DETERMINATION OF QUALITY OF DRINKING WATER

FROM DIFFERENT SOURCES
Monitoring for parameters such as Heterotropic Plate Count, Coliform/fecal coliform and
E. coli count is an approach of ensuring the quality of drinking water.
Total coliforms are a group of bacteria that are naturally found on plants and in soil,
water and in the intestine of humans and warm-blooded animals. Coliform bacteria are not
pathogenic, they are widespread in the environment, they can be used as operational tools to
determine the efficacy of drinking water treatment system from source-to-tap. Within the
coliforms are group of slightly heat resistant fecal coliform varieties and E. coli is of interest
since when present it indicates that resent fecal contamination has occurred with the possibility
of accompanying enteric pathogens like the enterohaemoragic strains of E. coli 0157.
MATERIALS
Culture (24-hour broth or slant) Equipment

Escherichia coli Biosafety Cabinet
Enterobacter aerogenes Incubator, 350C
Water bath, 44.50C
Media Sterile pipettes and pipette aid
Lauryl Tryptose Broth
Brilliant Green Lactose Bile Broth Miscellaneous supplies
EC medium Bacticinerator
Bactopeptone Diluents Inoculating loop and needle
Tryptone water Sterile tubes
Kovac’s reagent Vortex mixer
PROCEDURE
1. Follow the guidelines for proper collection and storage of water samples.
2. Collect water from two different sources. A minimum volume of 100 mL of water
should be collected for testing.
a. If the water sample is to be taken from a distribution-system, select
from a service pipe directly connected with the main and not from a
storage tank. Open tap fully and let water run to waste for 2 to 3
minutes. Reduce water flow to permit filling of bottle to about 250mL
b. In sampling from a mixing faucet, remove faucet attachments such as
screen or splash guard. Disinfect mouth of the faucet by running hot
water for 2 mins or by wiping 70% alcohol. Collect sample as
indicated above.
c. If the sample is to be taken from a well fitted with a hand pump, pump
water to waste for about 5 min. before collecting sample. If the well is
equipped with a mechanical pump, collect sample from a tap on the
discharge. If there is no pumping machinery, collect a sample directly
from the well by means of a sterilized bottle fitted with a weight at the
base; take care to avoid contaminating samples by any surface scum.
3. Analyzed the two samples for Heterotrophic Plate Count. Total coliform/fecal
coliform count and E. coli count to determine the potability of water from the two
water sources. Use the sample workeheets below.

ANNEX D
HETEROTROPHIC PLATE COUNT
Sample Worksheet
Sample Name: _____________________

Date received: ___________________
Sample Code: ______________________ Time received: ___________________
Result: Date analysis started: ___________________
Time started: ___________________
CFU/ml : __________________________ Date analysis finished: ___________________
Actual Volume Plate Counts Formula:

of Sample in
A B
dish CFU/ml = average colonies counted_____
1.0 ml Actual Volume of sample in dish,ml
0.1 ml
0.01 ml
0.001 ml
Analyzed By: __________________
Checked By: __________________
Date: _________________

ANNEX E
TOTAL COLIFORM COUNT, FECAL COLIFORM COUNT E. coli COUNT
Sample Worksheet
Date received: _________________
Sample Name: ______________________ Time received: _________________
Date analysis started: _________________
Sample Code: _______________________
Time started: _________________
Results: Date analysis finished: _________________
TOTAL COLIFORM COUNT , MPN/ 100 ml : ___________

FECAL COLIFORM COUNT , MPN/ 100 ml : ___________
E. coli COUNT , MPN/ 100 ml : ___________
Confirmatory Phase for Confirmatory Phase Completed
Tubes Presumptive test
Coliforms for Fecal Coliforms test for E. coli
(20 ml (LST broth)
(BLGB Broth) (EC Medium) (Indole test)
each)
24hr 48hr 24hr 48hr 24hr 48hr
1
2
3
4
5
MPN Index/100ml
Completed
Tubes Confirmatory Phase for Confirmatory Phase
Presumptive test test for E.
(10 ml Coliforms for Fecal Coliforms (EC
(LST Broth) coli
each) (BLGB Broth) Medium)
(Indole test)
1 24hr 48hr 24hr 48hr 24hr 48hr
2
3
4
5
6
7
8
9
10
MPN Index/100ml
Analyzed By: _____________________________ Checked By: __________________________

Date: _______________________________
ANNEX F
MPN Index and 95% Confidence Limits for all combinations of positive and
negative results when five 20 ml portions are used.
No. of tubes 95% Confidence
giving positive Limits (Exact)
MPN Index/
reaction out of Lower Higher
100 ml
five
(20 ml each)
0 < 1.1 - 3.5
1 1.1 0.051 5.4
2 2.6 0.40 8.4
3 4.6 1.0 13
4 8.0 2.1 23
5 >8.0 3.4 -
MPN Index and 95% Confidence Limits for all combinations of positive and
negative results when ten 10 ml portions are used.
No. of tubes giving 95% Confidence Limits
MPN Index/ 100
positive reaction (Exact)
out of ten ml
(10ml each) Lower Upper
0 < 1.1 -- 3.4
1 1.1 0.051 5.9
2 2.2 0.37 8.2
3 3.6 0.91 9.7
4 5.1 1.6 13
5 6.9 2.5 15
6 9.2 3.3 19
7 12 4.8 24
8 16 5.8 34
9 23 8.1 53
10 >23 13 --
Source:
Standard Methods for the Examination of Water and Wastewater. APHA-AWWA. 2005. 21st ed.

MPN Index and 95% Confidence Limits of Positive results when five tubes are
used per dilution (10ml, 1.0 ml, 0.1 ml)
Confidence MPN Index/ Confidence
Combination MPN Index/ Limits Combination Limits
100ml
of Positives 100ml of Positives
Low High Low High
0 0 0 < 1.8 -- 6.8 4 0 3 25 9.8 70
0 0 1 1.8 0.090 6.8 4 1 0 17 6.0 40
0 1 0 1.8 0.090 6.9 4 1 1 21 6.8 42
0 1 1 3.6 0.70 10 4 1 2 26 9.8 70
0 2 0 3.7 0.70 10 4 1 3 31 10 70
0 2 1 5.5 1.8 15 4 2 0 22 6.8 50
0 3 0 5.6 1.8 15 4 2 1 26 9.8 70
1 0 0 2.0 0.10 10 4 2 2 32 10 70
1 0 1 4.0 0.70 10 4 2 3 38 14 100
1 0 2 6.0 1.8 15 4 3 0 27 9.9 70
1 1 0 4.0 0.71 12 4 3 1 33 10 70
1 1 1 6.1 1.8 15 4 3 2 39 14 100
1 1 2 8.1 3.4 22 4 4 0 34 14 100
1 2 0 6.1 1.8 15 4 4 1 40 14 100
1 2 1 8.2 3.4 22 4 4 2 47 15 120
1 3 0 8.3 3.4 22 4 5 0 41 14 100
1 3 1 10 3.5 22 4 5 1 48 15 120
1 4 0 10 3.5 22 5 0 0 23 6.8 70
2 0 0 4.5 0.79 15 5 0 1 31 10 70
2 0 1 6.8 1.8 15 5 0 2 43 14 100
2 0 2 9.1 3.4 22 5 0 3 58 22 150
2 1 0 6.8 1.8 17 5 1 0 33 10 100
2 1 1 9.2 3.4 22 5 1 1 46 14 120
2 1 2 12 4.1 26 5 1 2 63 22 150
2 2 0 9.3 3.4 22 5 1 3 84 10 220
2 2 1 12 4.1 26 5 2 0 49 24 150
2 2 2 14 5.9 36 5 2 1 70 33 170
2 3 0 12 4.1 26 5 2 2 94 34 230
2 3 1 14 5.9 36 5 2 3 120 36 250
2 4 0 15 5.9 36 5 2 4 150 58 400
3 0 0 7.8 2.1 22 5 3 0 79 22 220
3 0 1 11 3.5 23 5 3 1 110 34 250
3 0 2 13 5.6 35 5 3 2 140 52 400
3 1 0 11 3.5 26 5 3 3 170 70 400
3 1 1 14 5.6 36 5 3 4 210 70 400
3 1 2 17 6.0 36 5 4 0 130 36 400
3 2 0 14 5.7 36 5 4 1 170 58 400
3 2 1 17 6.8 40 5 4 2 220 70 440
3 2 2 20 6.8 40 5 4 3 280 100 710
3 3 0 17 6.8 40 5 4 4 350 100 710
3 3 1 21 6.8 40 5 4 5 430 150 1100
3 3 2 24 9.8 70 5 5 0 240 70 710
3 4 0 21 6.8 40 5 5 1 350 100 1100
3 4 1 24 9.8 70 5 5 2 540 150 1700
3 5 0 25 9.8 70 5 5 3 920 220 2600
4 0 0 13 4.1 35 5 5 4 1600 400 4600
4 0 1 17 5.9 36 5 5 5 >1600 700 --
4 0 2 21 6.8 40

ANNEX G
Standard Methods of Detection and Values for Microbiological Quality

Parameters Methods of Value Units of Point of Compliance
Determination Measurement
Total Coliform Multiple Tube < 1.1 MPN/100ml  Service reservoirs
Fermentation  Water treatment works
Technique (MTFT)  Consumer’s taps
Chromogenic Absent  Refilling stations
Substrate test <1.1 MPN/100ml  Water Haulers
(Presence-Absence)  Water Vending
machines
Membrane Filter (MF) <1 Total coliform
Technique colonies/ 100 ml
Compliance to Total Coliform
(a) For water systems analyzing at least 40 samples per  Consumer’s taps
month, no more than 5% of the monthly sample may
be positive for total coliform
(b) For water systems analyzing fewer than 40 samples
per month, no more than one (1) sample per month
may be positive for total coliform
At least 95% of standard samples taken in each year  Service reservoirs
from each reservoir are total coliform negative
No standard sample taken each month should exceed  Water treatment works
maximum allowable value specified in the above  Refilling stations
 Water haulers
 Water vending machines
Fecal Coliform Multiple Tube < 1.1 MPN/100ml  Service reservoirs
Fermentation  Water treatment works
Technique (MTFT)  Consumer’s taps
Chromogenic  Refilling stations
Substrate test <1.1 MPN/100ml  Point Sources (Level 1)
(Presence-Absence)  Water Haulers
 Water Vending
Membrane Filter (MF) <1 Fecal coliform
Machines
Technique colonies/ 100 ml
Heterotrophic  Pour plate < 500 CFU/ml  Service reservoirs
Plate Count  Spread plate  Water treatment works
 Membrane  Consumer’s taps
 Refilling stations
filter technique
 Water Vending
Machines
Source:
Philippine National Standards for Drinking Water 2007
AO 2007-0012 by the Department of Health

ANNEX H
TRAINING SCHEDULE
Number of Hours
Day(s) Content
Lecture Laboratory
Welcome ceremony
0.5 Leveling of Expectations
Overview of the training
Topic 1 Water microbiology
1
1 Topic 2 Microbial analysis of water
Workshop No.1: Media Preparation & Quality
1
Control
Pre/post lab discussion for Heterotrophic
1.5
Plate Count
1.5 Method 1: Heterotropic Plate Count
0.5 Internal quality control
Workshop No. 2:
1.5  sample collection and handling
 dilution, inoculation and plating
2
Pre/post lab for Coliform/fecal coliform
1
determination
Method 2: Multiple-tube fermentation
3
technique
0.5 Pathogens significant in water
Laboratory observation: Confirmatory test for
1 0.5
E. coli & use of rapid test kit.
3
Reading, reporting and interpreting results.
1 3
Use of PNS standards for water
0.5 Wrap-up, feedback and evaluation
Total 18 hours

ANNEX I
Powerpoint Handouts
ANNEX J
Curriculum Vitae


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Food and Nutrition Research Institute

Department of Science and Technology

2 Training in Microbiological Analysis of Water

Topic 1 Introduction to Water Microbiology………………………….…….....…..6

METHOD 1 Heterotrophic Plate Count ……………….…………….…18

APPENDIX A WORKSHOP No. 1 Preparation and quality control of

3 Training in Microbiological Analysis of Water

Duration: Thirty (30) minutes

Specific: After the session, the participants must be able to

Content: Introduction / Overview of the Course

This training on microbiological analysis of water covers introduction to the

(b) enumerate and understand each test parameter included in water

(e) enumerate the laboratory requirements (e.g. equipment, facilities,

(f) conduct microbiological analysis of water in the laboratory; and

(g) demonstrate improvement in techniques on microbiological analysis of

Training Schedule (Annex E)

Resource Persons / Trainers: see attached curriculum vitae

5 Training in Microbiological Analysis of Water

Duration: One (1) hour

Specific: After the session, the participants should be able to:

Training Method: Lecture with visuals and discussion

Content: Water Microbiology

Clean Water is important to everyone however, still a significant number of

Over the years, there is still considerable number of outbreaks found to be

6 Training in Microbiological Analysis of Water

Contagious diseases caused by pathogenic bacteria, viruses and parasites are

7 Training in Microbiological Analysis of Water

American Public Health Association – American Water Works Association

Lechavallier, M. & Buckley, M. (2007). Clean Water: What is acceptable microbial

8 Training in Microbiological Analysis of Water

Philippines Department of Health. (9 Mar 2007). Philippine National Standards

World Health Organization. (2011). Chapter 5: Surveillance. Guidelines for

World Health Organization. (2011). Chapter 7:Microbial Aspects. Guidelines for

9 Training in Microbiological Analysis of Water

Duration: One (1) hour lecture, One (1) hour laboratory

Workshop no. 1 – Preparation and quality control of culture media.

Content: Microbiological Analysis of water

In assessing the microbial quality of water, it is very important that laboratory

It is also significant to learn the basic techniques found in the Compendium of

10 Training in Microbiological Analysis of Water

When examination will be delayed, it is particularly important to record the

11 Training in Microbiological Analysis of Water

6. Computation and Reporting of Results

In the conduct of microbiological analyses of water, validity of analytical data are

3. Laboratory Equipment and Instrumentation

12 Training in Microbiological Analysis of Water

 SOPs should describe in detail all laboratory operations

7. Analytical Quality Control Procedures

9. Documentation and Recordkeeping

13 Training in Microbiological Analysis of Water

Downes, F. & Ito, K. (2001). Compendium of Methods for the Microbiological

Philippines Accreditation Office (December, 2009). Supplementary Requirements for

Philippines Department of Health. (March 9, 2007). Philippine National Standards for

14 Training in Microbiological Analysis of Water

Duration: Fifteen (15) hours of lecture and laboratory

General: To discuss the techniques and quality control procedures employed

Observation training in the Food Analytical Service Laboratory on microbiological

Method 1 – Heterotrophic Plate Count: Pour plate Method

Workshop No. 2 – Determination of quality of drinking water from different

Laboratory supplies, Culture media/reagents, equipment for microbiological

15 Training in Microbiological Analysis of Water

16 Training in Microbiological Analysis of Water

Method 1 – Heterotrophic Plate Count

Analyzed By: ___ Checked By: